建構帶有輪狀病毒蛋白質基因之重組腺病毒

Abstract

中文摘要 輪狀病毒(Rotavirus)屬呼腸孤病毒科(Reoviridae)，為世界各地嬰幼兒病毒性急性腸胃炎的首要病因，造成嬰幼兒頗高的死亡率。因此，研發有效的輪狀病毒疫苗確為當務之急。 輪狀病毒顆粒由三層蛋白質殼構成。最外層為VP7、VP4，為分型之主要依據，具高度免疫性，刺激免疫細胞產生中和性抗體，為許多輪狀病毒疫苗研發針對之目標。VP6為構成中層蛋白質殼的主要成分，具高度抗原性及免疫生成性。NSP4為非結構性蛋白質，與病毒在細胞內的組裝有關，被認為是引發腹瀉症狀之腸毒素。文獻指出，針對VP6 或NSP4所產生之抗體，雖不具中和性但是卻可以產生保護的效果及減輕腹瀉症狀。加上二者在輪狀病毒的結構及功能上極為重要，較VP7、VP4更具保守性，故本研究之主要目標為建構同時帶有VP6 及NSP4基因之重組腺病毒期能達到加成之效果。此外，亦建構帶有VP7、VP4基因之重組腺病毒做為未來小鼠實驗用以對照之。 腺病毒載體為目前廣泛用於表現外來基因的一種系統。已有許多疫苗研究以其為載體。本研究選用腺病毒載體，將VP6及NSP4殖入同一穿梭載體中，與腺病毒基因同時送入大腸桿菌株BJ5183中，進行同源重組反應，產生同時具VP6 及NSP4基因之重組腺病毒基因。另外， VP7及VP4基因亦以同樣方式分別建構。將此三種帶有輪狀病毒基因之腺病毒基因轉染293A細胞，產生重組腺病毒rADV/VP4-Mu、rADV/VP7及rADV/VP6–NSP4。以之感染293A細胞株，聚合酶連鎖反應偵測目標基因，並進行螢光染色確認腺病毒蛋白質表現，輪狀病毒目標蛋白質表現則以西方墨點法、間接免疫螢光染色進行分析。陰性對照組為不帶有外來基因之腺病毒rADV/control。 目前已確定三種重組腺病毒皆可於293A細胞中進行複製，腺病毒Hexon蛋白質表現，可由螢光染色中明顯觀察到。目標蛋白質表現方面，VP6蛋白質表現較為明顯，但表現量則較預期為低，表現之細胞數目也遠較腺病毒感染之細胞數目少；NSP4則尚未觀察到其表現。VP7及VP4蛋白質部分，雖可於螢光染色下觀察到微弱的表現，但不甚明顯，表現的細胞比例也很低。必須先確認穿梭載體本身蛋白質表現無誤，再進一步觀察細胞感染重組腺病毒之後，目標基因之mRNA表現量及蛋白質表現量。

Abstract Rotaviruses, which form one genus of the family Reoviridae, are now recognized as the most important cause of severe viral gastroenteritis in infant and pre-school children. Because of the widespread nature of rotavirus disease, a safe and effective rotavirus vaccine is urgently needed, particularly in developing countries. The currently available live, attenuated oral rotavirus vaccine is promising, but improved vaccines are still needed. Recombinant adenovirus (rADV) vectors have received considerable attention for gene therapy because of their high transduction efficiency, infection of both dividing and resting cells of many cell types, including antigen-presenting cells, a feature that has called much attention upon them both as vaccine delivery vehicles. This study reports construction of rADV carrying a single gene, either VP7 or VP4 gene, and rADV carrying both VP6 and NSP4 genes of G9P[8] rotavirus. First, the plasmids carrying rotavirus gene, pShuttle-CMV/VP4, pShuttle- CMV/VP7 and pShuttle/VP6-NSP4, were constructed and co-transformed with adenovirus gene ( pAdEasy-1 ) into the E. coli BJ5183. After homologous recombination, the plasmid was purified and transfected into 293A cell line which could provide E1 protein that is necessary for rADV replication. The expression of rADV ( rADV/VP4-Mu, rADV/VP7 and rADV/VP6-NSP4 ) carrying the target gene was evaluated by polymerase chain reaction ( PCR ), western blotting and Immuno-fluorescence staining ( IFA ). rADV carrying only pShuttle-CMV（rADV/control）was used as the control. For confirmation of rADV carrying rotavirus gene after replication in 293A cell, DNA extracted from the infected cell was subjected to PCR. At present, the expression of the target protein could not be detected by western blotting. More experiments should be done to see if the target protein had been expressed in 293A cell and what made the differences in expression between adenovirus protein and target protein。